Antidiabetic 2-substituted-5&#39; -O- (1-Boranotriphosphate) adenosine derivatives

ABSTRACT

2-Substituted-5′-O-(1-boranotriphosphate)adenosine derivatives having at position 2 a radical R1 selected from the group consisting of H; halogen; O-hydrocarbyl; S-hydrocarbyl; NR3R4; and hydrocarbyl optionally substituted by halogen, CN, SCN, NO2, OR3, SR3 or NR3R4; wherein R3 and R4 are each independently H or hydrocarbyl or R3 and R4 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from oxygen, nitrogen and sulfur, and pharmaceutically acceptable salts or diastereoisomers thereof or a mixture of diastereoisomers, are useful for treatment of type 2 diabetes.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to new antidiabetic compounds and, inparticular, to novel 2-substituted-5′-O-(1-boranotriphosphate)-adenosinederivatives which are potent and selective insulin secretagogues.

Pathophysiology of Diabetes Mellitus

Diabetes mellitus is one of the most prevalent chronic diseases in theWestern world, affecting up to 5% of the population. It is aheterogenous group of disorders characterized by a chronic hyperglycemiawith additional abnormalities in lipid and protein metabolism. Thehyperglycemia results from defects in insulin secretion, insulin action,or a combination of both. In addition to its chronic metabolicabnormalities, diabetes is associated with long-term complicationsinvolving various organs, especially the eyes, nerves, blood vessels,heart and kidney, which may result in blindness, amputations,cardiovascular disease and end stage renal disease. The two major formsof diabetes are classified as type 1 and type 2. Type 2 diabetes,previously termed non-insulin-dependent diabetes mellitus (NIDDM), isthe most prevalent form of the disease, affecting approximately 95% ofpatients with diabetes.

Type 2 Diabetes Mellitus

The development of diabetic complications appears to be related to thechronic elevation of blood glucose. There is no current cure fordiabetes, however, effective glycemic control can lower the incidence ofdiabetic complications and reduce their severity.

Type 2 diabetes appears to be a complex polygenic disease in whichinsulin resistance and relative insulin deficiency coexist. Thus,improvement of insulin secretion is a major therapeutic goal. Thedeficiency of insulin release expresses itself not only by the absenceof first-phase insulin response to glucose, but also by a globalreduction in the magnitude of insulin release to 10-20% of the normalsecretory capacity (Cerasi, 1992).

Treatment of Hyperglycemia in Type 2 Diabetes Mellitus

Patients with type 2 diabetes are treated with various oral antidiabeticagents, insulin injections, or a combination of both. The currentlyavailable oral antidiabetic drugs are targeted at either reducingperipheral insulin resistance, increasing insulin secretion from thepancreatic beta-cell, or slowing the absorption of carbohydrates fromthe intestine.

Approximately half of the patients with type 2 diabetes are treated withoral agents, a considerable proportion of them with agents thatstimulate insulin secretion. The choice of insulin secretagogues islimited to the sulfonylureas and related compounds (“glinides”), whichelicit insulin secretion by binding to a regulatory subunit of membraneATP-sensitive potassium channel, inducing its closure (Lebovitz, 1994).Two types of agents are used to attenuate peripheral insulin resistance:the biguanide metformin and the thiazolidinedione analogues (Edelman,1998). The α-glucosidase inhibitor, pseudotetrasaccharide acarbose, isused to slow intestinal absorption of carbohydrates.

Sulfonylureas have several undesired effects in addition to possiblelong-term adverse effect on their specific target, the pancreaticbeta-cell. These side-effects include the risk of hypoglycemia due tostimulation of insulin secretion at low glucose concentrations, thedifficulty of achieving normal glycemia in a significant number ofpatients, the 5-10% per year secondary failure rate of adequate glycemiccontrol, and possible negative effects on the cardiovascular system(Lebovitz, 1994; Leibowitz and Cerasi, 1996; Brady and Terzic, 1998).

P2-Receptors

P2-receptors (P2-Rs) are membrane proteins that lead to inhibitory orexcitatory effects upon binding ADP, ATP or, in some subtypes, UTP(Bhagwat and Williams, 1997; King et al., 1998). A distinction was madebetween G-protein-coupled receptors and ligand-gated-ion-channelreceptors as the basis for the separation of P2-Rs into two broadclasses, P2Y and P2X, respectively (Abbracchio and Burnstock, 1994).P2-Rs are important targets for novel drug development for a variety ofpathophysiological conditions (Chan et al., 1998; Boarder and Hourani,1998; Barnard et al., 1997; Inoue, 1998; Abbracchio, 1996). Moreover,the large heterogeneity of P2-R subtypes in different tissues opens thepossibility of developing selective organ or tissue-specific P2-Rtargeted drugs.

The presence of P2-Rs of the P2Y subtype on pancreatic beta cells iswell documented (Loubatières-Mariani et al., 1979; Chapal andLoubatières-Mariani, 1981; Bertrand et al., 1987; Bertrand et al.,1991). The activation of pancreatic P2-Rs by extracellular ATP andstructural analogues results in stimulation of insulin secretion.Structure-activity relationships of the latter analogues have beeninvestigated (Chapal et al., 1997). The pharmacological properties andphysiological relevance of P2 receptors of the insulin-secreting cellhave been reviewed elsewhere (Petit et al., 1996; Loubatières-Mariani etal., 1997; Petit et al., 2001). A recent report suggests that inaddition to P2Y-Rs, functional P2X-Rs are also present on pancreaticbeta cells. However, whereas P2X-Rs augment insulin secretion at low,non-stimulating glucose levels, P2Y-Rs amplify insulin secretion only atstimulating glucose concentrations and do not affect, in contrast tosulfonylureas, the potassium conductance of the plasma membrane (Petitet al., 1998). The mechanism whereby P2Y-R agonists enhanceglucose-induced insulin release may involve the cyclic AMP/ProteinKinase A signaling pathway (Petit et al., 2000), which has been reportedto increase the effectiveness of the K⁺ _(ATP) channel-independentaction of glucose (Yajima et al., 1999). This coupling mechanism ofbeta-cell P2Y receptors is supported by the glucose-dependent insulinresponse induced by P2Y-Rs selective ligands.

P2Y-R Ligands as Potential Antidiabetic Drugs

Various P2-R selective ligands have been shown to increase insulinsecretion and decrease glycemia in vivo (Ribes et al., 1988;Hillaire-Buys et al., 1993). It was found that 2-methylthio-ATPstimulated insulin release and slightly decreased glycemia in the dog;however, to avoid its rapid breakdown into adenosine, this ATP analoguewas injected directly to the pancreatico-duodenal artery (Ribes et al.,1988). Adenosine 5′-O-(2-thio)diphosphate [ADP-β-S], which is stable toenzymatic hydrolysis, was administered either intravenously or orally torat and dog (Hillaire-Buys et al., 1993). In fed rats, ADP-β-S evoked asustained insulin response with a reduction of glycemia. In-vivoexperiments performed in conscious dogs have shown that this substancewas effective after oral administration, transiently increasinginsulinemia and reducing glycemia (Hillaire-Buys et al., 1993). It wasalso shown that the activation of P2Y-Rs was functionally effective inthe pancreas of diabetic animals (Hillaire-Buys et al., 1992; Tang etal., 1996). Moreover, it was recently reported that P2Y-R activationcould amplify glucose-induced insulin release from human pancreaticisolated islets (Fernandez-Alvarez et al., 2001).

Taken together, the data summarized above support the concept that P2Y-Ragonists may be considered as novel insulin-releasing compounds withpotential interest for the treatment of type 2 diabetes.

Identification of Potent, Stable and Subtype Selective P2Y-R Ligands

Almost all current synthetic P2-receptor agonists are modifications ofthe ATP or UTP pharmacophore. The purine (pyrimidine) ring system, theribose moiety, or the triphosphate chain are modified at one or morepositions (Fischer, 1999). Previously, we have reported the synthesis ofpotent and subtype selective P2-R-agonists (Fischer et al., 1993;Burnstock et al., 1994; Boyer et al., 1995; Boyer et al., 1996; Fischeret al., 1999). One series of these analogues represents ATP derivativesbearing a long thioether substitution at C-2 position (Fischer et al.,1993; Burnstock et al., 1994; Boyer et al., 1995; Fischer et al., 1999).Apparently, this substitution renders the molecule stable to enzymatichydrolysis (Zimmet et al., 1993). Moreover, it increases the potency ofthe molecules as P2Y-Rs ligands two to five orders of magnitude comparedwith ATP (Fischer et al., 1993; Burnstock et al., 1994; Boyer et al.,1995; Boyer et al., 1996).

2-Thioether-5′-O-(1-thiotriphosphate) Adenosine Derivatives as PotentialInsulin Secretagogues

In a previous study, we have synthesized novel P2Y-R ligands,2-thioether-5′-O-(1-thiotriphosphate)adenosine, 2-RS-ATP-α-S,derivatives (Fischer et al., 1999), as potential insulin secretagogues.The effects of the novel analogues on insulin secretion and pancreaticflow rate were evaluated on isolated and perfused rat pancreas. A highincrease, up to 500%, in glucose-induced insulin secretion was due tothe addition of 2-hexylthio-ATP-α-S in the nM concentration range, whichrepresents 100 fold enhancement of potency relative to ATP. Furthermore,these compounds are highly potent P2Y₁-R-ligands in turkey erythrocytesand exhibit relative enzymatic stability regarding pancreatic type IATPDase (Fischer et al., 1999). In addition, these compounds are highlychemically stable under physiological conditions and even underconditions simulating gastric juice acidity (Hillaire-Buys et al.,2001). However, their poor selectivity for the insulin-secreting cell,illustrated by their ability to induce vascular effects at insulinsecreting concentrations, made these derivatives a priori not suitablefor drug development as potential antidiabetics, since vascular eventsare the major pathophysiological complications of the disease.

SUMMARY OF THE INVENTION

It has now been found, according to the present invention, that certain2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivatives, hereinidentified as 2-R-ATP-α-B, act through P2Y(ATP)-receptors, present inthe membrane of pancreatic beta cells, as insulin secretagogues withhigh efficacy and potency, enhancing insulin secretion up to 900%, atthe nM concentration range, under slightly stimulatory glucoseconcentration.

The present invention thus relates to new compounds of the formula:

wherein

-   -   R₁ is selected from the group consisting of H; halogen;        O-hydrocarbyl; S-hydrocarbyl; NR₃R₄; and hydrocarbyl optionally        substituted by halogen, CN, SCN, NO₂, OR₃, SR₃ or NR₃R₄; wherein        R₃ and R₄ are each independently H or hydrocarbyl or R₃ and R₄        together with the nitrogen atom to which they are attached form        a saturated or unsaturated heterocyclic ring optionally        containing 1-2 farther heteroatoms selected from oxygen,        nitrogen and sulfur;    -   R₂ is H or hydrocarbyl, and    -   M⁺ represents the cation of a pharmaceutically acceptable salt,        or a diastereoisomer thereof or a mixture of diastereoisomers.

The compounds above are useful for enhancing insulin secretion andtreatment of type 2 diabetes.

Thus, in another embodiment, the invention relates to a pharmaceuticalcomposition, particularly for the treatment of type 2 diabetes,comprising at least one2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivative of theinvention, together with a pharmaceuticaly acceptable carrier ordiluent.

In a further embodiment, the invention relates to a method for enhancinginsulin secretion and treatment of type 2 diabetes which comprisesadministering to an individual in need thereof an effective amount of atleast one 2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivativeof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of increasing concentrations of the isomer A ofthe compound herein identified as 2-methylthio-ATPαB (VK 38A), oninsulin secretion from isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose. Theconcentrations are indicated in insert, with the number of independentexperiments (n) in each condition. The mean insulin output (ng/min) attime 45 min ranged between 10.43±3.89 and 14.37±0.47 according to theexperimental set. Each point represents the mean with SEM shown byvertical lines.

FIG. 2 shows the effects of increasing concentrations of VK 38A on thepancreatic flow rate from the isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose, at abaseline flow rate (time 45 min) of 2.5 mL/min. The concentrations areindicated in insert, with the number of independent experiments in eachcondition. Each point represents the mean with SEM shown by verticallines.

FIG. 3 shows the effects of increasing concentrations of isomer B of2-methylthio-ATPαB (VK 38B), on insulin secretion from the isolated ratpancreas perfused with a Krebs-bicarbonate buffer solution containing8.3 mmol/L glucose. The concentrations are indicated in insert, with thenumber of independent experiments in each condition. The mean insulinoutput (ng/min) at time 45 min ranged between 9.62±3.14 and 14.18±2.95according to the experimental set. Each point represents the mean withSEM shown by vertical lines.

FIG. 4 shows the effects of increasing concentrations of VK 38B on thepancreatic flow rate from the isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose, at abaseline flow rate (time 45 min) of 2.5 mL/min. The concentrations areindicated in insert, with the number of independent experiments in eachcondition. Each point represents the mean with SEM shown by verticallines.

FIG. 5 shows the effects of increasing concentrations of the isomer A ofthe compound herein identified as 2-chloro-ATPαB (VK 44A), on insulinsecretion from the isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose. Theconcentrations are indicated in insert, with the number of independentexperiments in each condition. The mean insulin output (ng/min) at time45 min ranged between 8.75±1.56 and 9.80±0.45 according to theexperimental set. Each point represents the mean with SEM shown byvertical lines.

FIG. 6 shows the effects of increasing concentrations of VK 44A on thepancreatic flow rate from the isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose, at abaseline flow rate (time 45 min) of 2.5 mL/min. The concentrations areindicated in insert, with the number of independent experiments in eachcondition. Each point represents the mean with SEM shown by verticallines.

FIG. 7 shows the effects of increasing concentrations of the isomer A ofthe compound herein identified as ATP-α-B (VK 39A), on insulin secretionfrom the isolated rat pancreas perfused with a Krebs-bicarbonate buffersolution containing 8.3 mmol/L glucose. The concentrations are indicatedin insert, with the number of independent experiments in each condition.The mean insulin output (ng/min) at time 45 min ranged between12.32±2.62 and 16.27±2.38 according to the experimental set. Each pointrepresents the mean with SEM shown by vertical lines.

FIG. 8 shows the effects of increasing concentrations of VK 39A on thepancreatic flow rate from the isolated rat pancreas perfused with aKrebs-bicarbonate buffer solution containing 8.3 mmol/L glucose, at abaseline flow rate (time 45 min) of 2.5 mL/min. The concentrations areindicated in insert, with the number of independent experiments in eachcondition. Each point represents the mean with SEM shown by verticallines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivatives of theformula shown hereinbefore.

As used herein, the term “halo” includes fluoro, chloro, bromo, andiodo, and is preferably chloro or bromo, most preferably chloro.

The term “hydrocarbyl” in any of the definitions of the differentradicals R₁-R₄ includes any saturated or unsaturated including aromatic,straight, branched or cyclic including polycyclic, radical containingcarbon and hydrogen such as, but not being limited to, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₀ cycloalkyl, aryl andar(C₀-C₈)alkyl.

The term “C₁-C₈ alkyl” typically means a straight or branchedhydrocarbon radical having 1-8 carbon atoms and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl,and the like. Preferred are C₁-C₆ alkyl groups, most preferably methyl.The terms “C₂-C₈ alkenyl” and “C₂-C₈ alkynyl” typically mean straightand branched hydrocarbon radicals having 2-8 carbon atoms and 1 doubleor triple bond, respectively, and include ethenyl, 3-buten-1-yl,2-ethenylbutyl, 3-octen-1-yl, and the like, and propynyl, 2-butyn-1-yl,3-pentyn-1-yl, and the like. C₂-C₆ alkenyl radicals are preferred. Theterm “C₃-C₁₀ cycloalkyl” means a cyclic or bicyclic hydrocarbyl groupsuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl,bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like. The term “aryl”denotes a carbocyclic aromatic radical such as phenyl and naphthyl andthe term “ar(C₁-C₈)alkyl” denotes an arylalkyl radical such as benzyland phenetyl.

When the radical R₁ is a O-hydrocarbyl or S-hydrocarbyl radical or ishydrocarbyl substituted by a O-hydrocarbyl or S-hydrocarbyl radical, thehydrocarbyl is preferably a C₁-C₆ alkyl, most preferably methyl, or anaryl, most preferably phenyl, or an aralkyl, most preferably benzyl,radical. In a most preferred embodiment, R₁ is SCH₃.

In the group NR₃R₄, R₃ and R₄ are each H or hydrocarbyl as defined aboveor form together with the N atom to which they are attached a saturatedor unsaturated, preferably a 5- or 6-membered, heterocyclic ring,optionally containing 1 or 2 further heteroatoms selected from nitrogen,oxygen, and sulfur. Such rings may be substituted, for example with oneor two C₁-C₆ alkyl groups. Examples of radicals NR₃R₄ include, withoutbeing limited to, amino, dimethylamino, diethylamino, ethylmethylamino,phenylmethylamino, pyrrolidino, piperidino, tetrahydropyridino,piperazino, morpholino, thiazolino, and the like.

Preferred compounds according to the invention are those wherein R₂ is Hand R₁ is Cl or, more preferably, 2-methylthio.

The invention encompasses the compounds themselves, a diastereoisomerthereof or a mixture of diastereoisomers as well as pharmaceuticallyacceptable salts thereof such as, but not limited to, compounds whereinM⁺ is Na⁺, K⁺, NH₄ ⁺ or the cation of an amine, particularly of atertiary amines e.g. N(R)₃H⁺, wherein R is preferably alkyl.

The compounds of the invention are prepared, for example, according tothe synthesis outlined in Scheme A hereinafter and exemplified in theexamples herein. Other compounds with different substituents areobtained by the same methods starting from suitable compounds orintroducing the desired groups during the synthesis by standard methodswell-known in the art.

The compounds of the invention are obtained as diastereoisomers whichcan be separated using a semipreparative reverse-phase Lichro CART250-10 column and isocratic elution [100 mM triethylammonium acetate(TEAA), pH 7 (A): MeOH (B), 84:16] with flow rate of 6 mL/min. Fractionscontaining the same isomer (similar retention time) are freeze-dried.The isomer with the shorter retention time is herein designated Isomer Aand the other, Isomer B. Isomers A of the compounds, and particularlyisomer A of the compound herein identified as 2-SMe-ATPαB, constitutepreferred embodiments of the invention.

In one preferred embodiment, the present invention relates to adiastereoisomer A of a compound of the invention, this diastereoisomer Abeing characterized by being the isomer with the shorter retention time(Rt) when separated from a mixture of diastereoisomers using asemipreparative reverse-phase Lichro CART 250-10 column and isocraticelution [100 mM triethylammonium acetate (TEAA), pH 7 (A): MeOH (B),84:16] with flow rate of 6 mL/min.

The 2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivatives ofthe invention are potent insulin secretagogues that target beta-cellP2Y-receptors, enhancing insulin secretion up to 900%, at the nMconcentration range, under slightly stimulatory glucose concentration.At these concentrations, the compounds of the invention have no or onlymild vascular side effects, in contrary to the most close compounds ofthe prior art, the P2Y-R ligands,2-thioether-5′-O-(1-thiotriphosphate)adenosine derivatives described byus previously (Fischer et al., 1999), which are also potent insulinsecretagogues but their poor selectivity for the insulin-secreting cell,illustrated by their ability to induce vascular effects at insulinsecreting concentrations, made these derivatives a priori not suitablefor drug development as potential antidiabetics, since vascular eventsare the major pathophysiological complications of the disease.

The compounds of the invention are P2Y receptor ligands with potentinsulin releasing action as well as with glucose-dependent amplifyingeffect on insulin secretion, which limit the risk of hypoglycemia andhave also limited vascular side effects, and are therefore suitable forthe treatment of type 2 diabetes. Thus, the present invention includeswithin its scope pharmaceutical compositions comprising, as an activeingredient, an effective amount of at least one2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivative of theinvention, or a pharmaceutically acceptable salt thereof, or adiastereoisomer or a mixture of diastereoisomers thereof, together witha pharmaceutically acceptable carrier or diluent.

Pharmaceutical compositions containing a compound of the presentinvention may be prepared by conventional techniques, e.g. as describedin Remington: The Science and Practice of Pharmacy, 19th Ed., 1995. Thecompositions may appear in conventional forms, for example capsules,tablets, solutions or suspensions.

The route of administration may be any route which effectivelytransports the active compound to the appropriate or desired site ofaction, the oral route being preferred. If a solid carrier is used fororal administration, the preparation may be tabletted, placed in a hardgelatin capsule in powder or pellet form or it can be in the form of alozenge. If a liquid carrier is used, the preparation may be in the formof a syrup, emulsion or soft gelatin capsule. Tablets, dragees, orcapsules having talc and/or a carbohydrate carrier or binder or the likeare particularly suitable for oral application. Preferable carriers fortablets, dragees, or capsules include lactose, corn starch, and/orpotato starch.

The invention further provides a method for treatment of a type 2diabetes patient, particularly for enhancing insulin secretion in saidpatient, which comprises administration of an effective amount of a2-substituted-5′-O-(1-boranotriphosphate)-adenosine derivative of theinvention. The dosage to be administered is in the range of 0.5-25 mg,preferably 1-20 mg, most preferably 1-10 mg, per day.

The invention will now be illustrated by the following non-limitativeExamples.

EXAMPLES

1. Chemistry

1.1 General Experimental Data

All air- and moisture-sensitive reactions were carried out inflame-dried, nitrogen flushed, two-neck flasks sealed with rubber septa,and the reagents were introduced with a syringe. Progress of reactionswas monitored by TLC on precoated Merck silica gel plates (60F-254).Column chromatography was performed with Merck silica gel 60 (230-400mesh). Compounds were characterized by nuclear magnetic resonance (NMR)using Brucker DPX-300, DMX-600, or AC-200 spectrometers. ¹H NMR spectrawere measured in D₂O, and the chemical shifts are reported in ppmrelative to HOD (4.78 ppm) as an internal standard. Nucleotides werecharacterized also by ³¹P NMR in D₂O, using 85% H₃PO₄ as an externalreference. All final products were characterized on an AutoSpec-E FISIONVG high-resolution mass spectrometer by chemical ionization. Nucleotideswere desorbed from a glycerol matrix under FAB (fast atom bombardment)conditions in low and high resolution. Primary purification of thenucleotides was achieved on an LC (Isco UA-6) system using a SephadexDEAE-A25 column, which was swelled in 1 M NaHCO₃ in the cold for 1 d.Final purification of the nucleotides and separation of thediastereoisomer pair was achieved on a HPLC (Merck-Hitachi) system usinga semipreparative reverse-phase (LiChrospher 60, RP-select-B) column.Conditions for LC and HPLC separation are described below.

1.2 Intermediates

2-Methylthio-adenosine was synthesized from 2-SH-adenosine as describedbefore (Methods in Enzymology, 1992, 215: 137-142). 2-SH-adenosine wasobtained from adenosine in three steps according to the procedurepreviously reported (J. Med. Chem. 1973, 16: 1381-1388; Chem. Pharm.Bull. 1977, 25: 1959-1969).

2-Chloro-adenosine was prepared in four steps from guanosine (Synthesis1982, 670-672) through2,6-Cl-9β-(2′,3′,5′-tri-O-acetyl)-D-ribofuranosylpurine (Can. J. Chem.1981, 59: 2601-2606), by treatment of the latter with NH₃ in EtOH in asealed ampule at 100° C. for 24 h.

1.3 Typical Procedure for the Preparation of 2′,3′-O-methoxymethylideneAdenosine Derivatives (Compounds 2 in Scheme A)

p-TsOH (2 mmol, 2 eq) was added to a dry adenosine derivative (1 mmol, 1eq) (Compound 1) in a two-neck flask under N₂, followed by addition ofdry DMF (4 mL). Then, trimethylorthoformate (50 eq) was added and theresulting solution was stirred at room temperature for 1 day. Themixture was cooled to 0° C. and Dowex MWA-1 (weakly basic anionexchanger, 6 eq) was added. Stirring continued at room temperature foradditional 3 h. The Dowex resin was filtered out in vacuo; the filtratewas concentrated under reduced pressure and coevaporated several timeswith MeOH to remove residual DMF. The residue was dissolved in CHCl₃ andextracted with saturated NaHCO₃. The organic phase was dried with Na₂SO₄and evaporated to give pure protected adenosine derivative 2.

According to this process the following compounds were prepared:

1.3.a) 2′,3′-O-Methoxymethylidene adenosine (compound 2 wherein R is H)was obtained from adenosine and trimethylorthoformate in 80% yield. ¹HNMR (DMSO-d₆, 300 MHz): δ 8.42, 8.38 (2s, H-8, 1H), 8.21, 8.20 (2s, H-2,1H), 7.42 (br.s, NH₂, 2H), 6.30, 6.20 (2d, J=3 Hz and J=2.8 Hz, H-1′,1H), 6.22, 6.11 (2s, CH—OCH₃, 1H), 5.53, 5.47 (2dd, J=2.8, 6 Hz and J=3,7 Hz, H-2′, 1H), 5.26, 5.20 (2t, J=5.5 Hz, OH-5′, 1H), 5.11, 5.03 (2dd,J=2.8, 6 Hz and J=3, 7 Hz, H-3′, 1H), 4.31, 4.24 (2dt, J=3, 5 Hz, H-4′,1H), 3.51-3.68 (m, H-5′, 2H), 3.40 (s, O—CH₃, 3H) ppm. ¹³C NMR (DMSO-d₆,300 MHz): δ 156.16 (C-6), 152.73 (CH-2), 148.88, 148.84 (C-4), 139.79,139.69 (CH-8), 119.04, 119.00 (C-5), 118.44, 116.93 (CH—OMe), 89.40,88.76 (CH-1′), 86.93, 85.90 (CH-2′), 83.47, 82.45 (CH-3′), 81.08, 80.72(CH-4′), 61.58, 61.32 (CH₂-5′), 51.87, 50.40 (OCH₃) ppm. MS CI/NH₃ m/z:310 (MH⁺).

1.3.b) 2-Methylthio-(2′,3′-O-methoxymethylidene)adenosine (compound 2wherein R is SCH₃) was obtained from 2-methylthioadenosine andtrimethylorthoformate in 75% yield. ¹H NMR (CDCl₃, 300 MHz): δ 7.96,7.93 (2s, H-8, 1H), 6.17, 5.91 (2d, J=3.6 Hz, J=3.9 Hz, H-1′, 1H), 6.05,5.97 (2s, CH—OMe, 1H), 5.47, 5.39 (2dd, J=3.9, 6 Hz, H-2′ and J=3.6, 7Hz, H-2′, 1H), 5.18-5.22 (m, H-3′, 1H), 4.55, 4.48 (2“q”, J=2.5 Hz,J=1.8 Hz, H-4′, 1H), 3.18-4.03 (m, H-5′, 2H), 3.46, 3.35 (2s, OCH₃, 3H),2.57, 2.56 (2s, S—CH₃, 3H) ppm. ¹³C NMR (CDCl₃, 300 MHz): δ 165.86(C-2), 154.31, 154.24 (C-6), 149.52 (C-4), 139.35 (C-8), 119.48, 117.74(CH—OMe), 117.41, 117.33 (C-5), 92.33, 92.03 (CH-1′), 87.41, 86.10(CH-2′), 83.87, 82.72 (CH-3′), 80.97, 80.73 (CH-4′), 62.79, 62.72(CH₂-5′), 52.95, 51.72 (O—CH₃), 14.47, 14.41 (S—CH₃) ppm. FAB (positivemode) m/z: 356.035 (MH⁺). HR FAB (positive mode) m/z: calcd forC₁₃H₁₇N₅O₅S (MH⁺) 356.1028, found 356.1038.

1.3.c) 2-Chloro-(2′,3′-O-methoxymethylidene)adenosine (compound 2wherein R is Cl) was obtained from 2-chloro-adenosine andtrimethylorthoformate in 81% yield. ¹H NMR (CDCl₃, 300 MHz): δ 7.70(H-8, 1H), 6.69 (s, NH₂, 2H), 6.16, 5.85 (2d, 3=3.6 Hz, 3=3.9 Hz, H-1′,1H), 6.03, 5.95 (2s, CH—OMe, 1H), 5.35-5.17 (2m, H-2′ and H-3′, 2H),4.53, 4.49 (2“br s”, H-4′, 1H), 4.02-3.8 (m, H-5′, 2H), 3.47, 3.32 (2s,OCH₃, 3H) ppm. ¹³C NMR (CDCl₃, 300 MHz): δ 163.54 (C-2), 156.45 (C-6),154.15 (C-4), 130.86 (C-8), 119.54, 117.67 (CH—OMe), 117.85 (C-5),92.71, 92.44 (CH-1′), 87.55, 85.94 (CH-2′), 83.98, 82.68 (CH-3′), 80.95,80.73 (CH-4′), 62.97, 62.88 (CH₂-5′), 53.04, 51.72 (O—CH₃) ppm. MSCI/NH₃ m/z: 344 (MH⁺). HRMS m/z: calcd for C₁₂H₁₄ClN₅O₅ 343.0683, found343.0671.

EXAMPLE 1 General Procedure for the Preparation of Derivatives ofadenosine-5′-O-(1-boranotriphosphate) (According to Scheme A)

Protected nucleoside 2 (0.5 mmol) was dissolved in dry CHCl₃ (7 mL) in aflame-dried, two-neck flask under N₂. (iPr)₂NEt (0.11 mL, 1.3 eq) wasadded at room temperature and the solution was stirred for 30 min. Themixture was cooled to 0° C. and [(iPr)₂N]₂PCl (148 mg, 1.1 eq),dissolved in CHCl₃ (2 mL), was slowly added with a syringe (step a), togive derivative 3. The resulting solution of derivative 3 was stirred at0° C. for 2 h followed by the addition of a 1 M solution of H₂P₂O₇ ⁻²(⁺HNBU₃)₂ in DMF (0.75 mL, 1.5 eq) (step b), to produce compound 4. Thissolution was kept at room temperature for additional 4 h and then cooledto 0° C. A 2 M solution of BH₃—SMe₂ complex in THF (2.52 mL, 10 eq) wasadded (step c). After 15 min of stirring at room temperature, deionizedwater (8 mL) was added and the resulting mixture was stirred for 1 h(step d) and then freeze-dried. Compound 6, obtained as a semisolid, wasdissolved in water and extracted with CHCl₃. The aqueous phase wasfreeze-dried and the resulting residue was applied on an activatedSephadex DEAE-A25 column (0-0.7 M NH₄HCO₃, total volume>2000 mL). Therelevant fractions were collected and freeze-dried; excess NH₄HCO₃ wasremoved by repeated freeze-drying with deionized water to yield compound6 as the tris ammonium salt. The methoxymethylidene protecting group wasremoved by acidic hydrolysis (10% HCl solution was added till pH 2.3 wasobtained). After 3 h at room temperature, the pH was rapidly raised to 9by the addition of NH₄OH solution (pH 11) and the solution was kept atroom temperature for 40 min (step e). The desiredadenosine-5′-O-(1-boranotriphosphate) derivative, herein designated“ATPαB derivative” (compound 7), was obtained after freeze-drying of thesolution. Final purification and separation of diastereoisomers of 7 wasachieved on a semipreparative HPLC column. The triethylammoniumcounterions were exchanged for Na⁺ by passing the pure diastereoisomerthrough Sephadex-CM C-25 column.

EXAMPLE 2 Preparation of adenosine-5′-O-(1-boranotriphosphate)

The title compound, herein identified as ATPαB [VK 39], was obtainedaccording to the procedure in Example 1 starting fromtetrabenzoyladenosine 8, in 19% yield.

EXAMPLE 3 Preparation of2-methylthioadenosine-5′-O-(1-boranotriphosphate)

The title compound, herein identified as 2-SMe-ATPαB [VK 38], wasobtained according to the procedure in Example 1 starting from2-thiomethyl-(2′,3′-O-methoxymethylidene)adenosine, in 38% yield.

EXAMPLE 4 Preparation of 2-chloroadenosine-5′-O-(1-boranotriphosphate)

The title compound, herein identified as 2-Cl-ATPαB [VK 44], wasobtained according to the procedure in Example 1 starting from2-chloro-(2′,3′-O-methoxymethylidene)adenosine, in 43% yield.

EXAMPLE 5 Reverse Phase HPLC Separation of Diastereoisomers ofadenosine-5′-O-(1-boranotriphosphate) Derivatives

The separation of diastereoisomers was accomplished using asemipreparative reverse-phase Lichro CART 250-10 column and isocraticelution [100 mM triethylammonium acetate (TEAA), pH 7 (A): MeOH (B),84:16] with flow rate of 6 mL/min. Fractions containing the same isomer(similar retention time) were freeze-dried. The excess buffer wasremoved by repeated freeze-drying with deionized water. The isomer withthe shorter retention time (Rt) is herein designated Isomer A and theother, Isomer B.

ATPαB, isomer A [VK 39A] (Rt 10.4 min), pH 6.5: ¹H NMR (D₂O, 200 MHz): δ8.62 (s, H-8, 1H), 8.25 (s, H-2, 1H), 6.16 (d, J=7 Hz, H-1′, 1H), 4.79(m, H-2′, 1H), 4.65 (m, H-3′, 1H), 4.42 (m, H-4′, 1H), 4.25 (m, H-5′,2H), 0.36 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz): δ 83.88 (m,P_(α)—BH₃), −9.42 (d, P_(γ)), −22.23 (t, P_(β)) ppm. FAB (negative mode)m/z: 526.162 (M⁴⁻+2H⁺+Na⁺). Isomer B [VK 39B](Rt 12.4 min), pH 6.5: ¹HNMR (D₂O, 200 MHz): δ 8.58 (s, H-8, 1H), 8.25 (s, H-2, 1H), 6.15 (d, J=7Hz, H-1′, 1H), 4.77 (m, H-2′, 1H), 4.56 (m, H-3′, 1H), 4.41 (m, H-4′,1H), 4.23 (m, H-5′, 2H), 0.36 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz):δ 84.5 (m, P_(α)—BH₃), −9.34 (d, P_(γ)), −22.2 (t, P_(β)) ppm. FAB(negative mode) m/z: 504.094.

2-SMe-ATPαB, isomer A [VK 38A](Rt 13.4 min) Na⁺ form, pH 7.5): ¹H NMR(D₂O, 300 MHz): δ 8.46 (s, H-8, 1H), 6.14 (d, J=5.3 Hz, H-1′, 1H), 4.69(dd, J=3.8, 4.9 Hz, H-3′, 1H), 4.38 (m, H-4′, 1H), 4.35, 4.14 (am, H-5′,2H), 2.59 (s, CH₃—S, 3H), 0.47 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz):δ 82.7 (m, P_(α)—BH₃), −6.5 (d, P_(γ)), −21.5 (t, P_(β)) ppm. FAB(negative mode) m/z: 550.172. Isomer B [VK 38B](Rt 15.6 min) (Na⁺ form,pH 7.5): ¹H NMR (D₂O, 300 MHz): δ 8.42 (s, H-8, 1H), 6.13 (d, J=5.6 Hz,1H), 4.86 (dd, J=5, 5.6 Hz, H-2′, 1H), 4.61 (dd, J=3.6, 5 Hz, H-3′, 1H),4.39 (q, J=3.6, 6 Hz, H-4′, 1H), 4.29 (ddd, J=2.9, 7.4, 11.8 Hz, H-5′,1H), 4.19 (ddd, J=2.9, 5.5, 11.8 Hz, H-5′, 1H), 2.59 (s, CH₃—S, 3H),0.46 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz) δ 83.9 (m, P_(α)—BH₃),−6.8 (d, P_(γ)), −21.6 (t, P_(β)) ppm. FAB (negative mode) m/z: 550.202.

2-Chloro-ATPαB, isomer A [VK 44A](Rt 10.2 min) (Na⁺ form, pH 7.5): ¹HNMR (D₂O, 300 MHz): δ 8.59 (s, H-8, 1H), 6.07 (d, J=5 Hz, H-1′, 1H),4.69 (dd, J=3.6, 4.5 Hz, H-3′, 1H), 4.41 (m, H-4′, 1H), 4.17, 4.37 (am,H-5′, 2H), 0.5 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz) δ 82.9 (m,P_(α)—BH₃), −6.01 (d, P_(γ)), −21.4 (t, P_(β)) ppm. FAB (negative mode)m/z: 559.023 (M⁴⁻+H⁺+Na⁺). Isomer B [VK 44B](Rt 12.6 min) (Na⁺ form, pH7.5): ¹H NMR (D₂O, 300 MHz): δ 8.54 (s, H-8, 1H), 6.04 (d, J=5.6 Hz,H-1′, 1H), 4.57 (dd, 3=3.5, 4.7 Hz, H-3′, 1H), 4.40 (m, H-4′, 1H), 4.30(ddd, J=2.6, 7.5, 11.5 Hz, H-5′, 1H), 4.18 (ddd, J=2.9, 5, 11.5 Hz,H-5′, 1H), 0.45 (m, BH₃, 3H) ppm. ³¹P NMR (D₂O, 200 MHz): δ 84.0 (m,P_(α)—BH₃), −6.4 (d, P_(γ)), −21.6 (t, P_(β)) ppm. FAB (negative mode)m/z: 559.765 (M⁴⁻+H⁺+Na⁺).

2. Pharmacology

The profile, efficacy and potency of the insulin response induced by thesynthetic ligands of the invention was evaluated in vitro in the modelof rat isolated pancreas. The effects of the compounds on pancreaticvascular resistance were also simultaneously recorded.

2.1 Methods

The effects of the compounds on insulin secretion and vascularresistance in the rat isolated and perfused pancreas were evaluated inthe presence of a slightly stimulating glucose concentration (8.3mmol/L).

Experiments were performed in vitro in isolated perfused pancreas frommale Wistar albino rats fed ad libitum and weighing 300-350 g. Thepancreas was completely isolated according to a technique previouslydescribed (Loubatières et al., Diabetologia, 1969, 5, 1-10) and perfusedthrough its own arterial system with a Krebs-Ringer bicarbonate buffercontaining 8.3 mmol/L glucose and 2 g/L bovine serum albumin. A mixtureof O₂ (95%) and CO₂ (5%) was bubbled through this medium at atmosphericpressure. The pH of the solution was 7.35. The preparation wasmaintained at 37.5° C. Each organ was perfused at a constant pressure(40-50 cm water) selected so as to produce a flow rate of 2.5 ml/min atthe start of the experiment; in these conditions, any change in the flowrate reflects a change in vascular resistance (Hillaire-Buys et al.,Eur. J. Pharmacol., 1991, 199, 309-314). A 30 min adaptation period wasallowed before the first sample was taken for insulin assay. A samplewas taken 15 min later, at time 45 min. Then, an infusion of ATPanalogues was performed during 30 min. The pancreatic flow rate wasrecorded and insulin was measured in the effluent fractions.

Insulin was assayed by the radioimmunological method of Herbert et al.(J. Clin. Endocrinol. Metab., 1965, 25, 1375-1384) using a purified ratinsulin as standard (Linco Research, St. Charles, Mo., USA) andanti-insulin serum (ICN Biochemicals, Miles, Puteaux, France). The assaysensitivity was 0.1 ng/ml. Insulin output from perfused pancreas isexpressed as ng/min and was determined by multiplying the hormoneconcentration in the effluent fraction by the flow rate.

Results are expressed as means±standard error of the mean (SEM). For thekinetics of insulin secretion and vascular flow rate, the results areexpressed as changes in relation to the value at time 45 min taken as100%. For the determination of the concentration-response relationship,the mean insulin output rate was calculated as follows: the area underthe curve for the drug infusion period divided by the number of minutes(AUC/30).

2.2 Results

The results below show that both diastereoisomers are pharmacologicallyactive but isomers A are more potent and selective than correspondingisomers B.

EXAMPLE 6 Effects of 2-methylthio-ATPαB (Isomers A and B)

The administration of the ATPαB derivative 2-methylthio-ATPαB, isomer A,identified as VK 38A, induced an immediate and concentration-dependentinsulin response in the range of 0.0015-5.0 μmol/L, as shown in FIG. 1.The increase in glucose-induced insulin release was first in a peak formfollowed by a second phase of sustained secretion (biphasic pattern),except for the lowest concentration (1.5 nmol/L) at which the druginduced a 230% transient monophasic insulin response. The maximal effectis obtained between 0.5 and 5.0 mmol/L and reaches approximately 900%(AUC for 30 min in % per min), with an EC₅₀ between 15 and 50 nmol/L.Concerning the vascular effects, no significant effect was observed till150 nmol/L; a slight and transient reduction in pancreatic flow rate(increased vascular resistance) was observed at 0.5, 1.5 and 5.0 μmol/L,reaching −6±3%, −10±3% and −8±6%, respectively (FIG. 2).

The administration of isomer B of 2-methylthio-ATPαB, identified as VK38B, induced an insulin response of similar pattern, although lesspotent than that induced by isomer A (FIG. 3). Moreover, in contrast toisomer A, VK 38B induced a clear and transient −27±5% reduction inpancreatic flow rate (increase in vascular resistance) from theconcentration of 15 nmol/L (FIG. 4).

EXAMPLE 7 Effects of 2-chloro-ATPαB (Isomer A)

The administration of isomer A of 2-chloro-ATPαB, identified as VK 44A,induced an insulin response which seems comparable to that of VK 38A(FIG. 5). However, it also induced a slight and sustained increase inpancreatic flow rate (decreased vascular resistance), reaching +8±3%,+16±5% and +12±5% at 0.015, 0.15 and 1.5 μmol/L, respectively (FIG. 6).

EXAMPLE 8 Effects of ATPαB (Isomer A)

The administration of the parent compound ATPαB (isomer A), identifiedas VK 39A, induced a biphasic insulin response clearly less potent thanthat of VK 38 A (FIG. 7); it also induced a slight and sustainedvascular response, increasing the pancreatic flow rate by +7±3% and+10±3% at 0.5 and 5.0 μmol/L, respectively (FIG. 8).

EXAMPLE 9 Glucose-Dependence of the Insulin Response Triggered by VK 38Ain the Isolated Rat Pancreas

The rat isolated pancreas is perfused in vitro with a physiologicalmedium containing different concentrations of glucose. VK 38A is addedduring 20 minutes at 20 nmol/L. Insulin response is determined by thearea under the concentration-time curve during VK 38A administration andis expressed as mean±sem. The results are shown in Table 1. TABLE 1Glucose concentration Insulin response (mmol/L) (ng/min) 2.8 0.97 ± 0.035.0 2.68 ± 0.52 8.3 33.12 ± 1.75 

EXAMPLE 10 The Effect of VK 38 A on Glycemia In Vivo

Normal (non diabetic) Wistar rats were treated with a single oral doseof VK 38A (0.2 mg/kg) or placebo (vehicle) administered just before aglucose tolerance test, in a cross-over experimental design with a 7-daywash-out period. Glucose (2 g/kg) was administered by intraperitonealinjection. Blood samples were taken before and 10, 20, 30, 60 minutesafter glucose load to measure glycemia. The area under the curve ofplasma glucose concentrations (in percent of baseline values) wascalculated. Results in four animals are given in Table 2 (baselineglucose concentrations were 1.26±0.02 and 1.26±0.04 g/L in control andtreated animals, respectively). TABLE 2 AUC (VK38A- Animal AUC (control)treated) 1 173 158 2 224 201 3 147 134 4 197 176

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1. A 2-substituted-5′-O-(1-boranotriphosphate)adenosine compound of theformula:

wherein R₁ is selected from the group consisting of H; halogen;O-hydrocarbyl; S-hydrocarbyl; NR₃R₄; and hydrocarbyl optionallysubstituted by halogen, CN, SCN, NO₂, OR₃, SR₃ or NR₃R₄; wherein R₃ andR₄ are each independently H or hydrocarbyl or R₃ and R₄ together withthe nitrogen atom to which they are attached form a saturated orunsaturated heterocyclic ring optionally containing 1-2 furtherheteroatoms selected from oxygen, nitrogen and sulfur; R₂ is H orhydrocarbyl, and M⁺ represents the cation of a pharmaceuticallyacceptable salt, or a diastereoisomer thereof or a mixture ofdiastereoisomers.
 2. A compound according to claim 1, wherein R₁ ishydrocarbyl, O-hydrocarbyl or S-hydrocarbyl, and said hydrocarbyl isselected from a saturated or unsaturated, including aromatic, straight,branched or cyclic, including polycyclic, radical containing carbon andhydrogen.
 3. A compound according to claim 1, wherein R₁ is NR₃R₄, andR₃ and R₄ are each independently H or hydrocarbyl or R₃ and R₄ togetherwith the nitrogen atom to which they are attached form a saturated orunsaturated heterocyclic ring optionally containing 1-2 furtherheteroatoms selected from oxygen, nitrogen and sulfur.
 4. A compoundaccording to claim 2, wherein said hydrocarbyl is selected from C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₀ cycloalkyl, aryl orar(C₁-C₈)alkyl.
 5. A compound according to claim 4, wherein saidhydrocarbyl is selected from C₁-C₆ alkyl, phenyl or benzyl.
 6. Acompound according to claim 5, wherein R₁ is S-C₁-C₆ alkyl.
 7. Acompound according to claim 6, wherein R₁ is S—CH₃. 8.2-Methylthioadenosine-5′-O-(1-boranotriphosphate).
 9. A compoundaccording to claim 1, wherein R₁ is halogen.
 10. A compound according toclaim 9, wherein R₁ is chloro or bromo.
 11. A compound according toclaim 10, wherein R₁ is chloro. 12.2-Chloroadenosine-5′-O-(1-boranotriphosphate).
 13. The diastereoisomer Aof a compound according to claim 1, characterized by being the isomerwith the shorter retention time (Rt) when separated from a mixture ofdiastereoisomers using a semipreparative reverse-phase Lichro CART250-10 column and isocratic elution [100 mM triethylammonium acetate(TEAA), pH 7 (A): MeOH (B), 84:16] with flow rate of 6 mL/min.
 14. Thediastereoisomer A of 2-methylthioadenosine-5′-O-(1-boranotriphosphate)(Rt 13.4 min).
 15. A pharmaceutical composition comprising at least one2-substituted-5′-O-(1-boranotriphosphate)adenosine derivative accordingto claim 1, or a pharmaceutically acceptable salt thereof, or adiastereoisomer or a mixture of diastereoisomers thereof, together witha pharmaceutically acceptable carrier or diluent.
 16. A pharmaceuticalcomposition according to claim 15 comprising2-methylthioadenosine-5′-O-(1-boranotriphosphate).
 17. A pharmaceuticalcomposition according to claim 15 comprising at least onediastereoisomer of said2-substituted-5′-O-(1-boranotriphosphate)adenosine derivative.
 18. Apharmaceutical composition according to claim 17 comprising adiastereoisomer A of said2-substituted-5′-O-(1-boranotriphosphate)adenosine derivative.
 19. Apharmaceutical composition according to claim 18 comprising thediastereoisomer A of 2-methylthioadenosine-5′-O-(1-boranotriphosphate).20. A pharmaceutical composition according to claim 15 for enhancementof insulin secretion and treatment of type 2 diabetes.
 21. Apharmaceutical composition according to claim 20 for oraladministration.
 22. A method for treatment of type 2 diabetes whichcomprises administering to a diabetic patient in need an effectiveamount of a compound of claim
 1. 23. A method for treatment of type 2diabetes which comprises administering to a diabetic patient in need aneffective amount of 2-methylthioadenosine-5′-O-(1-boranotriphosphate).